The role of integrins in embryonic development, inflammation, tissue repair, and tumorigenesis depends in large part on their ability to organize the cytoskeleton and to transmit to the cell interior signals from the extracellular matrix. The alpha6-beta4 integrin is a receptor involved in the adhesion of epithelial, neuroepithelial and endothelial cells to basement membranes. This receptor has a structure different from that of all the other known integrins, since the intracellular portion of beta4 subunit is unusually large (ca. 110 kD) and contains toward its C- terminus two pairs of type III fibronectin-like modules. While most integrins cluster in adhesion plaques upon binding to extracellular ligand, alpha6-beta4 concentrates in hemidesmosomes. In addition, in contrast to some integrins which are downregnlated upon neoplastic transformation, alpha6-beta4 is expressed at higher levels in invasive carcinomas of breast, pancreas and skin than it is in corresponding benign adenomas and normal tissues. These findings raise the possibility that the alpha6-beta4 integrin has unique cytoskeletal and signaling interactions. Our preliminary studies indicate that a specific region of beta4 cytoplasmic domain, comprising the first two type III repeats and the segment between the second and third repeat, may promote the assembly of alpha6-beta4 at hemidesmosomes by interacting with cytoskeletal component(s). The same region is phosphorylated on tyrosine upon Epidermal Growth Factor stimulation, it is cleaved by the calcium dependent protease calpain in some tissues, and it is interrupted by inserted extra-sequences in the variant forms of beta4 generated by alternative splicing of mRNA. Therefore, it is possible that phosphorylation, proteolytic cleavage and alternative splicing represent mechanisms for regulating the intracellular interactions of alpha6-beta4. We here propose to analyze the structure, function and regulation of the intracellular domain of beta4. The amino acid sequences involved in the interaction of beta4 tail with hemidesmosomal components will be defined by introducing targeted mutations in the beta4 cDNA and by expressing the mutant forms in hemidesmosome forming cells. The functional significance of beta4 phosphorylation will be examined by identifying the tyrosine residue(s) affected and by comparing the ability of wild-type and mutant unphosphorylatable receptor to interact with the cytoskeleton and to bind extracellular ligand. Finally, molecules interacting with the large cytoplasmic domain of beta4 will be identified and characterized by methods of blot overlay, chemical crosslinking, affinity chromatography, and expression cloning. It is expected that these studies will increase our understanding of the intracellular interactions of integrins.